A method and apparatus for performing melting curve analyses of nucleic acids on a microarray is described herein.
Microarray technology evolved in the late 1980s and early 1990s from nucleic acid hybridization assays such as the Southern Blot and Northern Blot. The microarray technique is essentially a high throughput competitive hybridization with many different nucleic acid sequences of probes attached to an array surface, and many different sequences of target DNA. This technique has been adapted to a variety of applications, but some of the most common are gene expression profiling and single nucleotide polymorphism (SNP) analysis. In gene expression assays, mRNA is extracted from cells, converted to cDNA and simultaneously stained with fluorescent dyes before being hybridized to array chips containing complementary probes. The chips are allowed to dry, scanned via an array reader, and gene expression differences are calculated via software. In this procedure, the greater the level of gene expression, the more the fluorescent target DNA binds to the probes, and the stronger is the fluorescent signal. In SNP analysis, cDNA synthesis is omitted because genomic DNA is extracted from cells, is restriction digested, and then hybridized to chips (solid supports). In SNP analysis the array contains at least four probes for each SNP with a different base at the expected SNP, with a different base at the expected SNP site. The array reading process is the same for SNP analysis as for gene expression but some binding to all probes may be observed, and the correct SNP is measured by determining which probe has the most DNA binding.
Microarray techniques allow for high sample throughput. However, known microarray techniques exhibit reduction in accuracy and repeatability in comparison to high accuracy low throughput techniques. Like all other nucleic acid competitive binding assays, known microarray techniques suffer from the tendency of target DNA to bind the wrong probe, a condition known as promiscuous binding or mismatch. Promiscuous binding can be reduced by selecting the optimal hybridization temperature for a given sequence. However, because the array system may have several thousand different probe sequences on one array, it is practically impossible to optimize hybridization temperatures for all probes simultaneously. Rather an average hybridization temperature for all probes is calculated. But even under optimized conditions promiscuous binding still occurs. It is believed that this tendency, as well as lack of standardization in probe selection, is responsible for much of the error remaining in microarray analysis.
Although microarray techniques have produced valuable results, the standard application requires that results be validated using more accurate and expensive methods such as a real-time polymerase chain reaction (PCR). For gene expression experiments, the fold differences in expression of a single gene can vary as much as several fold between array results and real-time PCR validation results.
Methods for improving microarrays should deal directly with promiscuous binding issues. High accuracy, low throughput techniques such as real-time PCR, which is an enzyme-based test, are more accurate because of temperature-controlled hybridization of PCR primers. Therefore, double stranded product is only made if correct hybridization of primers to template takes place. During a typical real-time PCR experiment, it is possible to check the quality of the PCR reaction by examining the melting curve of the PCR product.
Melting curve analysis involves the use of heat to break the hydrogen bonds holding double stranded nucleic acids (most often DNA) together so that the double stranded form melts apart (“dissociates”) into two single stranded products. The amount of heat required to melt DNA is dependent on the length, cytosine-guanine (C-G) content, and the complementarily of the double stranded form. In practice, special dyes such as SYBR Green I are among those used to monitor the exact temperature at which melting occurs. SYBR Green fluoresces 1000 times more when intercalated between double stranded DNA verses floating free in solution. As the temperature is raised, a large reduction in fluorescence indicates the melting of double stranded DNA and is usually depicted in a graph of fluorescence verses temperature. If mispriming takes place during real-time PCR reactions, the presence of a second PCR product with a different melting temperature would be evident, indicating that something went wrong with the reaction, and the experiment must be repeated under better conditions.